1.

Plasmid DNA

Contents What is Plasmid DNA? Competent Cells and Transformation Growth and Culture of Bacteria Bacterial culture media and antibiotics Storage of E. coli strains Growth of E. coli cultures Lysis of Bacterial Cells for Plasmid Purification Isopropanol Precipitation of DNA A Guide to Analytical Gels Pouring an agarose gel Running an agarose gel Visual analysis of the gel Agarose Gel Analysis of Plasmid DNA Quantification of DNA Restriction Endonuclease Digestion of DNA Ligation of DNA 2 2 4 4 6 7 9 10 11 11 13 14 15 16 18 20

Plasmid DNA

What is Plasmid DNA?

Bacterial plasmids are closed circular molecules of double-stranded DNA that range in size from 1 to >200 kb. They are found in a variety of bacterial species, where they behave as additional genetic units inherited and replicated independently of the bacterial chromosome. However, they rely upon enzymes and proteins provided by the host for their successful transcription and replication. Plasmids often contain genes that code for enzymes that can be advantageous to the host cell in some circumstances. The encoded enzymes may be involved in resistance to, or production of, antibiotics, resistance to toxins found in the environment (e.g., complex organic compounds), or the production of toxins by the bacteria itself. Once purified, plasmid DNA can be used in a wide variety of downstream applications such as sequencing, PCR, expression of proteins, transfection, and gene therapy. This chapter describes common plasmid DNA procedures, including how to make and transform competent cells, how to culture and handle plasmidcontaining cells, and how to purify and quantify plasmid DNA.

Competent Cells and Transformation

Protocol 1. Preparation of competent E. coli Cells that have the ability to take up DNA (from a variety of sources) are termed competent. Several techniques exist to prepare competent cells and one such technique for preparing competent E. coli is given below. Note: Cells prepared using this protocol are not suitable for electroporation. Materials

Protocol 1

E. coli cells in glycerol stock vial LB medium LB-agar plates

Appropriate selective antibiotics TFB1 buffer TFB2 buffer

For buffer and media compositions, see Bacterial Culture Media and Buffers, page 92. 1. Remove a trace of E. coli cells from the glycerol stock vial with a sterile toothpick or inoculating loop, and streak it out on LB-agar plates containing an appropriate concentration of the relevant selective antibiotic(s) (see Antibiotics, page 5). If the host strain has already been cultured and stored at 28C (cultures can be stored at 28C for up to 3 months without any significant loss of viability), streak out bacteria from those stocks. 2. Incubate at 37C overnight. 3. Pick a single colony and inoculate 10 ml LB medium containing relevant antibiotic(s). Grow overnight at 37C.
protocol continues overleaf

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Protocol 1. Continued 4. Add 1 ml overnight culture to 100 ml prewarmed LB medium containing the relevant antibiotic(s) in a 500 ml flask, and shake at 37C until an OD600 of 0.5 is reached (approximately 90120 min). 5. Cool the culture on ice for 5 min, and transfer the culture to a sterile, round-bottom centrifuge tube. 6. Collect the cells by centrifugation at low speed (5 min, 4000 x g, 4C). 7. Discard the supernatant carefully. Always keep the cells on ice. 8. Resuspend the cells gently in cold (4C) TFB1 buffer (30 ml for a 100 ml culture) and keep the suspension on ice for an additional 90 min. 9. Collect the cells by centrifugation (5 min, 4000 x g, 4C). 10. Discard the supernatant carefully. Always keep the cells on ice. 11. Resuspend the cells carefully in 4 ml ice-cold TFB2 buffer. 12. Prepare aliquots of 100200 l in sterile microcentrifuge tubes and freeze in liquid nitrogen or a dry-iceethanol mix. Store the competent cells at 70C.

Tip

Several commercial kits (e.g., the QIAGEN PCR Cloning competent cells for optimized transformation.

plus

Kit for cloning of PCR products) offer ready-to-use

Protocol 2. Transformation of competent E. coli cells Transformation is the process in which plasmid DNA is introduced into a bacterial host cell. Several methods exist for transformation of bacterial cells, one of which is given below. Materials

Protocol 2

Competent E. coli cells (see Protocol 1, above) SOC medium LB-agar plates

For buffer and media compositions, see Bacterial Culture Media and Buffers, page 92. 1. Transfer an aliquot of the DNA to be transformed (10 l or less) into a cold sterile 1.5 ml microcentrifuge tube, and keep it on ice. 2. Thaw an aliquot of frozen competent E. coli cells on ice. 3. Gently resuspend the cells and transfer 100 l of the cell suspension into the microcentrifuge tube with the plasmid DNA, mix carefully, and keep on ice for 20 min. 4. Transfer the tube to a 42C water bath or heating block for 90 s. 5. Add 500 l SOC medium to the cells and incubate for 6090 min at 37C.

Tip

Shaking increases transformation efficiency.

6. Plate out 50, 100, and 200 l aliquots on LB-agar plates containing the relevant antibiotic(s). Incubate the plates at 37C overnight until colonies develop.

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Positive control to check transformation efficiency Transform competent cells with 1 ng of a control plasmid containing an antibiotic resistance gene. Plate onto LB-agar plates containing the relevant antibiotic(s). Compare the number of colonies obtained with the control plasmid to the number obtained with the plasmid of interest to compare transformation efficiency. Negative control to check antibiotic activity Transform cells with 20 l of TE. Plate at least 200 l of the transformation mix on a single LB-agar plate containing the relevant antibiotic(s). An absence of colonies on the plates indicates that the antibiotic is active.

Growth and Culture of Bacteria*

Bacterial culture media and antibiotics
Liquid media Liquid cultures of E. coli can generally be grown in LB (Luria-Bertani) medium. Please note that a number of different LB broths are commonly used. We recommend using the LB composition given in Bacterial Culture Media and Buffers (page 92) to obtain the highest yields of plasmid DNA. Sterilizing media Sterilize liquid or solid media by autoclaving, using a pressure and time period suitable for the type of medium, bottle size, and autoclave type.

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It is advisable to autoclave liquid medium in several small bottles rather than in one large vessel, to avoid possible contamination of an entire batch. Fill bottles only 3/4 full with medium and loosen the caps before autoclaving to avoid hot medium boiling over. Tighten caps once the media is cool (<40C) to maintain sterility. Antibiotics and medium supplements such as amino acids are degraded by autoclaving. Antibiotics should be added to liquid medium immediately prior to use from stock solutions that have been filter-sterilized, distributed into aliquots, and stored in the dark at 20C (see Antibiotics, page 5).

* More extensive coverage of microbiological technique can be found in current manuals: see references 1 and 2, page 21.

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Solid media E. coli strains can generally be streaked and stored, for a short period of time, on LB plates containing 1.5% agar and the appropriate antibiotic(s). Preparation of LB-agar plates Prepare LB medium according to the composition given in Bacterial Culture Media and Buffers, page 92. Just before autoclaving, add 15 grams agar per liter and mix. After autoclaving, swirl the medium gently to distribute the melted agar evenly throughout the solution.

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Cool autoclaved agar medium to below 50C (when you can hold it comfortably) before adding heat-sensitive antibiotics and nutrients. Mix thoroughly before pouring. Pour plates in a laminar-flow hood or on a cleaned bench surface next to a Bunsen burner. Use 3035 ml medium per standard 90 mm petri dish. After pouring plates, any air bubbles may be removed by passing the flame of a Bunsen burner briefly over the surface.

Dry plates by removing the lids and standing the plates in a laminar-flow hood for 1 hour; with the covers slightly open in a 37C incubator for 30 minutes; or left upside down with lids on at room temperature overnight. Store plates inverted at 4C in a dark room or wrapped in aluminum foil to preserve light-sensitive antibiotics. Do not store for longer than 1 month as antibiotics may degrade. Label plates with the date and the antibiotic used.

Antibiotics Bacterial strains carrying plasmids or genes with antibiotic selection markers should always be cultured in liquid or on solid medium containing the appropriate selective agent. Lack of antibiotic selection can lead to loss of the plasmid carrying the genetic marker and potentially to selection of faster-growing mutants!

Tip Tip

Prepare stock solutions of antibiotics separately from batches of liquid or solid media, sterilize by filtration, aliquot, and store in the dark at 20C. Recommended stock and working concentrations for commonly used antibiotics are shown in Table 1. Before adding antibiotics to freshly autoclaved medium, ensure that the medium has cooled to below 50C.

Table 1. Concentrations of commonly used antibiotics Antibiotic Ampicillin (sodium salt) Chloramphenicol Kanamycin Streptomycin Tetracycline HCl Carbenicillin Stock solutions Concentration Storage 50 mg/ml in water 34 mg/ml in ethanol 10 mg/ml in water 10 mg/ml in water 5 mg/ml in ethanol 50 mg/ml in water 20C 20C 20C 20C 20C 20C Working concentration (dilution) 100 g/ml (1/500) 170 g/ml (1/200) 50 g/ml (1/200) 50 g/ml (1/200) 50 g/ml (1/100) 50 g/ml (1/1000)

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Storage of E. coli strains

There are different methods for storing E. coli strains depending on the desired storage time. Glycerol stocks and stab cultures enable long-term storage of bacteria, while agar plates can be used for short-term storage. When recovering a stored strain, it is advisable to check that the antibiotic markers have not been lost by streaking the strain onto an LB-agar plate containing the appropriate antibiotic(s).

Protocol 3. Preparation of glycerol stocks E. coli strains can be stored for many years at 70C in medium containing 15% glycerol. Prepare glycerol stocks of bacteria as follows: 1. Add 0.15 ml glycerol (100%) to a 2 ml screw-cap vial and sterilize by autoclaving.

Protocol 3

Tip

Vials of sterilized glycerol can be prepared in batches and stored at room temperature until required.

2. Add 0.85 ml of a logarithmic-phase E. coli culture (see Growth of E. coli cultures, page 7) to the vial of pre-sterilized glycerol.

3. Vortex the vial vigorously to ensure even mixing of the bacterial culture and the glycerol. 4. Freeze in an dry iceethanol bath or liquid nitrogen and store at 70C.

Tip Tip

Avoid repeated thawing and re-freezing of glycerol stocks as this can reduce the viability of the bacteria. For precious strains, storage of two stock vials is recommended.

Protocol 4. Preparation of stab cultures E. coli strains can also be stored for up to 1 year as stabs in soft agar. Stab cultures can be used to transport or send bacterial strains to other labs. Prepare stab cultures as follows: 1. Prepare and autoclave 0.7% LB agar (standard LB medium containing 7 g/liter agar) as described in Bacterial Culture Media and Buffers, page 92. 2. Cool the LB agar to below 50C (when you can hold it comfortably) and add the appropriate antibiotic(s). While still liquid, add 1 ml agar to a 2 ml screw-cap vial under sterile conditions, then leave to solidify. Vials of agar can be prepared in batches and stored at room temperature until required. 3. Using a sterile straight wire, pick a single colony from a freshly grown plate and stab it deep down into the soft agar several times. 4. Incubate the vial at 37C for 812 h leaving the cap slightly loose. 5. Seal the vial tightly and store in the dark, preferably at 4C.

Protocol 4

Tip

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Growth of E. coli cultures

Figure 1 shows the sequence of steps necessary to go from a stored stock of bacteria to a liquid culture for plasmid isolation. Bacterial stocks should always be streaked onto selective plates prior to use, to check that they give rise to healthy colonies carrying the appropriate antibiotic resistance. Stocks can potentially contain mutants arising from the cultures used to prepare them, or can deteriorate during storage. E. coli Handling
Glycerol stock (long-term storage 70C)

Bacteria streaked on agar plates (short-term storage 4C)

Stab culture (long-term storage 4C)

Freshly-streaked, well-isolated bacterial colonies

Liquid pre-culture (~8 h)

Liquid culture (1216 h)

Plasmid DNA preparation Figure 1. Essential steps for storage and handling of E. coli.

Protocol 5. Recovery of single colonies from stored cultures Plates of streaked bacteria can be sealed with Parafilm and stored upside-down at 4C for several weeks.

Protocol 5

Bacteria should always be streaked onto plates containing the appropriate antibiotic to ensure that selective markers are not lost. To obtain isolated colonies, streak an agar plate as follows: 1. Flame a wire loop, and cool on a spare sterile agar plate. 2. Using the wire loop, streak an inoculum of bacteria (from a glycerol stock, stab culture, or single colony on another plate) across one corner of a fresh agar plate, as shown in Figure 2. 3. Flame and cool the wire loop again. Pass it through the first streak and then streak again across a fresh corner of the plate. 4. Repeat again to form a pattern as in Figure 2. 5. Incubate the plate upside down at 37C for 1224 h until colonies develop. 6. Inoculate liquid cultures from a healthy, well-isolated colony, picked from a freshly streaked selective plate. This will ensure that cells growing in the culture are all descended from a single founder cell, and have the same genetic makeup.
3rd streak

Streaking Bacteria
1st streak 2nd streak

Tip

Culture volumes >10 ml should not be inoculated directly from a plate, but with a pre-culture of 25 ml diluted 1/500 to 1/1000.

Figure 2. Streaking bacteria on agar plates.

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E. coli growth curve The growth curve of an E. coli culture can be divided into distinct phases (Figure 3). Lag phase occurs after dilution of the starter culture into fresh medium. Cell division is slow as the bacteria adapt to the fresh medium. After 45 hours the culture enters logarithmic (log) phase, where bacteria grow exponentially. Cells enter stationary phase (~16 hours) when the available nutrients are used up. The cell density remains constant in this phase. Eventually the culture enters the phase of decline, where cells start to lyse, the number of viable bacteria falls, and DNA becomes partly degraded. Measuring cell density The growth curve of a bacterial culture can be monitored photometrically by reading the optical density at 600 nm (Figure 3). Note however that photometric measurements of cell density can vary between different spectrophotometers. E.coli Growth Curve
5.0 4.5 4.0

Cell density (OD600)

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 0 5 10 15 20

Tip Tip Tip

Calibrate your spectrophotometer by determining the number of cells per milliliter giving a particular OD600 reading. Plate serial dilutions of a culture on LB agar plates and calculate the number of cells per milliliter in the original culture. This is then set in relation to the measured OD600 value. High OD600 readings should be calculated by diluting the sample in culture medium to enable measurement in the linear range of 0.10.5 OD600.

Time (h)

Figure 3. Growth curve of E. coli in LB medium. Host strain: DH5; plasmid: pUC21. High OD600 readings were calculated by diluting the sample to enable photometric measurement in the linear range of 0.10.5 OD600

Another way of estimating the amount of cell harvest is to assess the pellet wet weight. Typically a 1 liter, overnight culture of E. coli within a cell density of 34 x 109 cells per milliliter corresponds to a pellet wet weight of approximately 3 grams.

Protocol 6. Preparation of bacteria for plasmid preps To prepare the bacterial culture for your plasmid prep, follow the steps below. 1. Prepare a starter culture by inoculating a single colony from a freshly grown selective plate into 210 ml LB medium containing the appropriate antibiotic. Grow at 37C for ~8 h with vigorous shaking (~300 rpm).

Protocol 6

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It is often convenient to grow the starter culture during the day so that the larger culture can be grown overnight for harvesting the following morning. Use a flask of at least 5 times the volume of culture to ensure sufficient aeration. Do not use a larger culture volume than recommended in the protocol, as use of too many cells will result in inefficient lysis and reduce the quality of the preparation. Growth for 1216 h corresponds to the transition from logarithmic into stationary growth phase (see Figure 3), when cell density is high (34 x 109 cells per ml) and RNA content of cells is low. Growth of cultures is dependent on factors such as host strain, plasmid insert and copy number, and culture medium. To determine the optimal harvesting time for a particular system, monitor the cell density and the growth of the culture by measuring the OD600 (see previous section).

2. Dilute the starter culture 1/500 to 1/1000 into a larger volume of selective LB medium.

3. Grow the culture at 37C with vigorous shaking (~300 rpm) for 1216 h.

4. Harvest the bacterial culture by centrifugation at 6000 x g for 15 min at 4C. Remove all traces of the supernatant. The cells are now ready for the lysis procedure.

Tip

The procedure may be stopped at this point and continued later by freezing the cell pellets obtained by centrifugation. The frozen cell pellets can be stored at 20C for several weeks.

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Lysis of Bacterial Cells for Plasmid Purification

Effective lysis of bacterial cells is a key step in plasmid isolation as DNA yield and quality depend on the quality of cell lysate used for the purification. Alkaline lysis Alkaline lysis is one of the most commonly used methods for lysing bacterial cells prior to plasmid purification (3, 4). Production of alkaline lysates involves four basic steps (Figure 4): 1. Resuspension Harvested bacterial cells are resuspended in TrisClEDTA buffer containing RNase A.
2. Lysis
NaOH/SDS

Alkaline Lysis Procedure

1. Resuspension
E. coli cell

3. Neutralization
KAc

Tip Tip Tip

Ensure that bacteria are resuspended completely leaving no cell clumps in order to maximize the number of cells exposed to the lysis reagents. Buffer volumes for alkaline lysis in QIAGEN plasmid purification procedures are optimized for particular culture volumes in LB medium. Do not use a culture volume larger than recommended in the protocol as this will lead to inefficient lysis and reduce the quality of the plasmid preparation. For large scale purification of low-copy plasmids, for which larger cultures volumes are used, it may be beneficial to increase the lysis buffer volumes in order to increase the efficiency of alkaline lysis and thereby the DNA yield.

4. Clearing of lysates
Centrifugation Filtration

Supernatant containing plasmid DNA

KDS precipitate containing genomic DNA + proteins

Plasmid DNA Chromosomal DNA

Filtrate containing plasmid DNA

Figure 4. Principle of alkaline lysis.

2. Lysis Cells are lysed with NaOH/SDS. Sodium dodecyl sulfate (SDS) solubilizes the phospholipid and protein components of the cell membrane, leading to lysis and release of the cell contents. NaOH denatures the chromosomal and plasmid DNA, as well as proteins. The presence of RNase A ensures that liberated cellular RNA is digested during lysis.

Tip Tip Tip

If after addition of lysis buffer (NaOH/SDS) the solution appears very viscous and is difficult to mix, this indicates excess biomass in the lysate step. This results in insufficient cell lysis and it is recommended to double the amount of lysis and neutralization buffers used. Avoid vigorous stirring or vortexing of the lysate as this can shear the bacterial chromosome, which will then copurify with the plasmid DNA. The solution should be mixed gently but thoroughly by inverting the lysis vessel 46 times. Do not allow the lysis to proceed for longer than 5 minutes. This is optimal for release of the plasmid DNA, while avoiding irreversible plasmid denaturation.

3. Neutralization The lysate is neutralized by the addition of acidic potassium acetate. The high salt concentration causes potassium dodecyl sulfate (KDS) to precipitate, and denatured proteins, chromosomal DNA, and cellular debris are coprecipitated in insoluble salt-detergent complexes. Plasmid DNA, being circular and covalently closed, renatures correctly and remains in solution.

Tip Tip

Precipitation can be enhanced by using chilled neutralization buffer and incubating on ice.

4. Clearing of lysates Precipitated debris is removed by either centrifugation or filtration, producing cleared lysates. QIAGEN lysate filtration technologies ensure removal of all precipitated material without centrifugation, considerably reducing plasmid purification time.

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Purification of plasmid DNA from cleared bacterial lysates was traditionally performed using cesium chloride (CsCl) ultracentrifugation. Today, a variety of commercially available plasmid purification kits offer easy procedures for different throughput requirements and applications.

Isopropanol Precipitation of DNA

Alcohol precipitation is commonly used for concentrating, desalting, and recovering nucleic acids. Precipitation is mediated by high concentrations of salt and the addition of either isopropanol or ethanol. Since less alcohol is required for isopropanol precipitation, this is the preferred method for precipitating DNA from large volumes. In addition, isopropanol precipitation can be performed at room temperature, which minimizes co-precipitation of salt that may interfere with downstream applications. This section provides hints on how to perform an effective isopropanol precipitation and to help ensure maximum recovery of DNA. The range of values given reflects protocol variation depending on the scale and type of preparation.

Protocol 7. Isopropanol precipitation procedure 1. Adjust the salt concentration if necessary, e.g., with sodium acetate (0.3 M, pH 5.2, final concentration) or ammonium acetate (2.02.5 M, final concentration). 2. Add 0.60.7 volumes of room-temperature isopropanol to the DNA solution and mix well.

Protocol 7

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Use all solutions at room temperature to minimize co-precipitation of salt. Centrifugation should be carried out at 4C to prevent overheating of the sample. (When precipitating from small volumes, centrifugation may be carried out at room temperature.) Marking the outside of the tube or uniformly orienting microcentrifuge tubes before centrifugation allows the pellet to be more easily located. Pellets from isopropanol precipitation have a glassy appearance and may be more difficult to see than the fluffy salt-containing pellets that result from ethanol precipitation. Care should be taken when removing the supernatant as pellets from isopropanol precipitation are more loosely attached to the side of the tube. Carefully tip the tube with the pellet on the upper side to avoid dislodging the pellet. For valuable samples, the supernatant should be retained until recovery of the precipitated DNA has been verified.
protocol continues overleaf

3. Centrifuge the sample immediately at 10,00015,000 x g for 1530 min at 4C.

4. Carefully decant the supernatant without disturbing the pellet.

5. Wash the DNA pellet by adding room-temperature 70% ethanol. This removes co-precipitated salt and replaces the isopropanol with the more volatile ethanol, making the DNA easier to redissolve.

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Protocol 7. Continued 6. Centrifuge at 10,00015,000 x g for 515 min at 4C.

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Centrifuge the tube in the same orientation as previously to recover the DNA in a compact pellet. The QIAprecipitator (QIAGEN) ensures recovery of precipitated DNA without centrifugation, considerably reducing plasmid purification time and eliminating the risk of DNA pellet loss. Do not overdry the pellet (e.g., by using a vacuum evaporator) as this will make DNA, especially high-molecular-weight DNA, difficult to redissolve. Choose an appropriate volume of buffer according to the expected DNA yield and the desired final DNA concentration. Use a buffer with a pH 8.0 for redissolving, as DNA does not dissolve easily in acidic buffers. (If using water, check pH.) Redissolve by rinsing the walls to recover all the DNA, especially if glass tubes have been used. To avoid shearing DNA do not pipet or vortex. High-molecular-weight DNA should be redissolved very gently to avoid shearing, e.g., at room temperature overnight or at 55C for 12 h with gentle agitation.

7. Carefully decant the supernatant without disturbing the pellet.

8. Air-dry the pellet for 520 min (depending on the size of the pellet).

9. Redissolve the DNA in a suitable buffer.

A Guide to Analytical Gels

This section is aimed at providing useful hints for effective gel analysis of nucleic acids. Firstly, the basic steps involved in pouring an agarose gel for DNA analysis are outlined. Subsequent sections look at loading and running the gel and visualization of the DNA. Principle of gel analysis Gels allow separation and identification of nucleic acids based on charge migration. Migration of nucleic acid molecules in an electric field is determined by size and conformation, allowing nucleic acids of different sizes to be separated. However, the relationship between the fragment size and rate of migration is non-linear, since larger fragments have greater frictional drag and are less efficient at migrating through the polymer. Agarose gel analysis is the most commonly used method for analyzing DNA fragments between 0.1 and 25 kb. Other specialized analytical gel methods exist for analyzing extremely large or small DNA molecules. Detailed information on all types of analytical gels can be found in current molecular biology manuals (1, 2).

Pouring an agarose gel

Agarose concentration The concentration of agarose used for the gel depends primarily on the size of the DNA fragments to be analyzed. Low agarose concentrations are used to separate large DNA fragments, while high agarose concentrations allow resolution of small DNA fragments (Table 2).

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Table 2. Concentration of agarose used for separating DNA of different sizes Agarose concentration (% w/v) 0.3* 0.5 0.7 1.0 1.2 1.5 2.0*
Adapted from references 1 and 2. * Most gels are run using standard agarose, although some special types of agarose are available for particular applications and for very high or low agarose concentrations. For example, low-melt agarose allows in situ enzymatic reactions and can be used for preparative gels.

DNA fragment range (kb) 560 130 0.812 0.510 0.47 0.23 0.052

Electrophoresis buffers The most commonly used buffers for agarose gel electrophoresis are TBE (Tris-borateEDTA) and TAE (Tris-acetateEDTA) (see Agarose Gel Electrophoresis Buffers for Analysis of DNA, page 93). Although more frequently used, TAE has a lower buffering capacity than TBE and is more easily exhausted during extended electrophoresis. TBE gives better resolution and sharper bands, and is particularly recommended for analyzing fragments <1 kb. The drawback of TBE is that the borate ions in the buffer form complexes with the cis-diol groups of sugar monomers and polymers, making it difficult to extract DNA fragments from TBE gels using traditional methods.

Tip

QIAGEN Systems for gel extraction utilize optimized buffers for efficient extraction of DNA fragments from both TBE and TAE gels.

Protocol 8. Pouring the gel 1. Prepare enough 1x running buffer both to pour the gel and fill the electrophoresis tank. 2. Add an appropriate amount of agarose (depending on the concentration required) to an appropriate volume of running buffer (depending on the volume of the gel tray being used) in a flask or bottle.

Protocol 8

Tip Tip Tip Tip

The vessel should not be more than half full. Loosely cover the vessel to minimize evaporation. Note: The cover should not be airtight. Always use the same batch of buffer to prepare the agarose as to run the gel, since small differences in ionic strength can affect migration of DNA.

3. Heat the slurry in a microwave or boiling water bath, swirling the vessel occasionally, until the agarose is dissolved. Ensure that the lid of the flask is loose to avoid buildup of pressure. Be careful not to let the agarose solution boil over as it becomes superheated. If the volume of liquid reduces considerably during heating due to evaporation, make up to the original volume with distilled water.

4. Cool the agarose to 5560C. Add ethidium bromide if desired (see page 14). 5. Pour the agarose solution onto the gel tray to a thickness of 35 mm. Insert the comb either before or immediately after pouring. Leave the gel to set (3040 min).

Tip Tip

Ensure that there is enough space between the bottom of the comb and the glass plate (0.51.0 mm) to allow proper formation of the wells and avoid sample leakage.

6. Carefully remove the comb and adhesive tape, if used, from the gel. Fill the tank containing the gel with electrophoresis buffer. Add enough buffer to cover the gel with a depth of approximately 1 mm liquid above the surface of the gel. If too much buffer is used the electric current will flow through the buffer instead of the gel.

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Running an agarose gel

Preparation of samples Agarose gel analysis with ethidium bromide staining allows detection of DNA amounts from as little as 20 ng up to 500 ng in a band (5 mm wide x 2 mm deep). Loading of larger amounts of DNA will result in smearing of the DNA bands on the gel.

Tip Tip Tip

Samples must always be mixed with gel loading buffer prior to loading (see below). Be sure that all samples have the same buffer composition. High salt concentrations will retard the migration of the DNA fragments. Ensure that no ethanol is present in the samples, as this will cause samples to float out of the wells.

Gel loading buffers and markers Gel loading buffer (see Agarose Gel Electrophoresis Buffers for Analysis of DNA, page 93) must be added to the samples before loading and serves three main purposes: 1. To increase the density of the samples to ensure that they sink into the wells. 2. To add color to the samples through use of dyes such as bromophenol blue, Orange G, or xylene cyanol, facilitating loading. 3. To allow tracking of the electrophoresis due to co-migration of the dyes with DNA fragments of a specific size. Molecular-weight markers should always be included on a gel to enable analysis of DNA fragment sizes in the samples. See Commonly Used DNA Markers in Agarose Gel Electrophoresis, page 93 for commonly used markers.

Protocol 9. Electrophoresis 1. Apply samples in gel loading buffer to the wells of the gel.

Protocol 9

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Prior to sample loading, rinse wells with electrophoresis buffer. Make sure that the entire gel is submerged in the running buffer. Once samples are loaded, do not move the gel tray/tank as this may cause samples to float out of the wells. Electrophoresis apparatus should always be covered to protect against electric shocks.

2. Connect the electrodes so that the DNA will migrate towards the anode (positive electrode). 3. Turn on the power supply and run the gel at 110 V/cm until the dyes have migrated an appropriate distance. This will depend on the size of DNA being analyzed, the concentration of agarose in the gel, and the separation required. Avoid use of very high voltages which can cause trailing and smearing of DNA bands in the gel, particularly with high-molecular-weight DNA. Monitor the temperature of the buffer periodically during the run. If the buffer becomes heated, reduce the voltage. Melting of an agarose gel during electrophoresis is a sign that the voltage is too high, that the buffer may have been incorrectly prepared or has become exhausted during the run.

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Visual analysis of the gel

Staining To allow visualization of the DNA samples, agarose gels are stained with an appropriate dye. The most commonly used dye is the intercalating fluorescent dye ethidium bromide, which can be added either before or after electrophoresis (see Table 3). Alternatives include commercial dyes such as SYBR Green.

Tip Tip

Stock solutions of ethidium bromide (generally 10 mg/ml) should be stored at 4C in a dark bottle or bottle wrapped in aluminum foil.

Addition of ethidium bromide prior to electrophoresis add ethidium bromide to a final concentration of 0.5 g/ml to the melted and subsequently cooled agarose, i.e., just before pouring the gel. Mix the agaroseethidium bromide solution well to avoid localized staining. Addition of ethidium bromide after electrophoresis soak the gel in a 0.5 g/ml solution of ethidium bromide (in water or electrophoresis buffer) for 3040 minutes.

Note: Ethidium bromide is a powerful mutagen and is very toxic. Wear gloves and take appropriate safety precautions when handling. Use of nitrile gloves is recommended as latex gloves may not provide full protection. After use, ethidium bromide solutions should be decontaminated as described in commonly used manuals (1, 2).

Tip Tip

Rinse the gel with buffer or water before examining it to remove excess ethidium bromide. Staining buffer can be saved and re-used.

Table 3. Comparison of ethidium bromide staining methods Addition of ethidium bromide prior to electrophoresis Faster and more convenient procedure Allows monitoring of migration throughout the procedure Requires decontamination of gel tanks and comb More ethidium bromide is required Electrophoretic mobility of linear DNA fragments is reduced by ~15% Visualization Ethidium bromideDNA complexes display increased fluorescence compared to the dye in solution. This means that illumination of a stained gel under UV light (254366 nm) allows bands of DNA to be visualized against a background of unbound dye. The gel image can be recorded by taking a Polaroid photograph or using a gel documentation system. Addition of ethidium bromide after electrophoresis Slower procedure requiring additional step Does not allow monitoring of migration during electrophoresis No decontamination of gel tanks and comb necessary Usually less ethidium bromide is required No interference with electrophoretic mobility

Tip Tip

UV light can damage the eyes and skin. Always wear suitable eye and face protection when working with a UV light source. UV light damages DNA. If DNA fragments are to be extracted from the gel, use a lower intensity UV source if possible and minimize exposure of the DNA to the UV light.

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Agarose Gel Analysis of Plasmid DNA

The main uses of agarose gels for plasmid DNA analysis are: Analysis of the Plasmid Purification Procedure
M 1 2 3 4 A B C D E M

Analysis of the size and conformation of nucleic acids in a sample Quantification of DNA (see page 16) Separation and extraction of DNA fragments

Analysis of a purification procedure Figure 5 shows how agarose gel electrophoresis can be used to analyze the nucleic acid content of samples taken during a plasmid purification procedure. The gel demonstrates successful plasmid purification using anion-exchange columns as well as some atypical results. M: Lambda DNA digested with HindIII. 1: Cleared lysate containing supercoiled (lower band) and open circular plasmid DNA (upper band) and degraded RNA (smear at the bottom of the gel). 2: Flow-through fraction containing only degraded RNA (the plasmid DNA is bound to the anion-exchange resin in the column). 3: Wash fraction to ensure that the resin in the column is cleared of RNA and other contaminants (plasmid DNA remains bound to the column). 4: Eluate containing pure plasmid DNA in supercoiled and open circular forms.

Figure 5. Agarose gel analysis of a plasmid purification procedure using QIAGEN anion-exchange tips. Samples were taken at different stages of the procedure. 2 l of each sample was run on a 1% agarose gel. M: lambda-HindIII markers.

Lanes AE illustrate some atypical results that may be observed in some preparations, depending on plasmid type and host strain. A: Supercoiled (lower band) and open circular form (upper band) of the high-copy plasmid, pUC18, with an additional band of denatured supercoiled DNA migrating just beyond the supercoiled form. B: Multimeric forms of supercoiled plasmid DNA (pTZ19) that may be observed with some host strains and should not be mistaken for genomic DNA. Multimeric plasmid DNA is easily distinguished from genomic DNA by restriction digestion. C: Linearized form of plasmid pTZ19 after restriction digestion with EcoRI. D: Sample contaminated with bacterial chromosomal DNA (uppermost band). E: EcoRI digestion of a sample contaminated with bacterial genomic DNA, which gives a smear above the plasmid DNA.

Tip

With large-constructs such as BAC, PAC, and P1 DNA, the supercoiled form migrates at a slower rate than the linear form. Furthermore, large-construct DNA >50 kb is often difficult to distinguish from genomic DNA by agarose gel analysis.

Gel extraction Agarose gels can be used for separation and extraction of DNA fragments, for example, a specific DNA fragment from a PCR or restriction digestion reaction.

Tip Tip Tip

Ensure that the percentage of agarose used for the gel allows good separation of DNA fragments for easy excision. Run agarose gels for DNA extraction at a low voltage. This will enable efficient separation of DNA bands without smearing, facilitating excision of the gel slice. Excise the fragment quickly under low-strength UV light to limit DNA damage.

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DNA fragments can be extracted quickly and efficiently from agarose gels using silica-gelbased purification. Silica-gelbased methods typically result in higher and more reproducible recoveries than other gel extraction methods, such as electroelution, and require no phenol extraction or ethanol precipitation. In a typical silica-gelbased purification procedure, the agarose gel slice is first solubilized. DNA is then bound to the silica-gel material in the presence of high concentrations of chaotropic salts. A wash step removes impurities, and DNA is then eluted in low-salt buffer.

Tip

QIAGEN offers three kits for silica-gelbased purification of differently sized DNA fragments from agarose gels, which differ in methodology and elution volumes: Purification of fragments between 70 bp and 10 kb in a spin-column format which can be used in a microcentrifuge or on a vacuum manifold. Purification of fragments between 70 bp and 4 kb in a spin-column format, in elution volumes of only 10 l. Purification of fragments between 40 bp and 50 kb using silica-gel particles.

Polyacrylamide gel electrophoresis (PAGE) As an alternative to agarose gel electrophoresis, polyacrylamide gels can be used for the analytical or preparative separation of small, double-stranded DNA fragments. This method is applicable to DNA fragments from 10 to 1000 bp. The resolution and capacity of polyacrylamide gels is higher than that of agarose gels. However, agarose gels are much easier to pour and run, and in the vast majority of cases deliver acceptable resolution. Protocols for PAGE of DNA can be found in standard molecular biology texts (1, 2).

Quantification of DNA
Reliable measurement of DNA concentration is important for many applications in molecular biology. Plasmid DNA quantification is generally performed by spectrophotometric measurement of the absorption at 260 nm, or by agarose gel analysis. In this section, we examine some critical factors for quantification, such as the effect of solvents, phenol, and RNA contamination on absorption. DNA quantification by spectophotometry Plasmid DNA concentration can be determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer using a quartz cuvette. For reliable DNA quantification, A260 readings should lie between 0.1 and 1.0. An absorbance of 1 unit at 260 nm corresponds to 50 g plasmid DNA per ml (A260 = 1 50 g/ml).* This relationship is only valid for measurements made at neutral pH, therefore, samples should be diluted in a low-salt buffer with neutral pH (e.g., TrisCl, pH 7.0). An example of the calculation involved in nucleic acid quantification when using a spectrophotometer is provided in Spectrophotometric Measurement of Nucleic Acid Concentration, page 91. When working with small amounts of DNA, such as purified PCR products or DNA fragments extracted from agarose gels, quantification via agarose gel analysis may be more effective (see DNA quantification by agarose gel analysis, page 17).

Tip

* Based on a standard 1 cm path length.

Plasmid DNA

If you will use more than one quartz cuvette to measure multiple samples, the cuvettes must be matched.

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Effects of solvents on spectrophotometric readings Absorption of nucleic acids depends on the solvent used to dissolve the nucleic acid (6). A260 values are reproducible when using low-salt buffer, but not when using water. This is most likely due to differences in the pH of the water caused by the solvation of CO2 from air. A260/A280 ratios measured in water also give rise to a high variability between readings (Figure 6) and the ratios obtained are typically <1.8, resulting in reduced sensitivity to protein contamination (6). In contrast, A260/A280 ratios measured in a low-salt buffer with slightly alkaline pH are generally reproducible. Effect of RNA contamination on spectrophotometric readings Plasmid DNA preparations can contain RNA contamination, for example, when the RNase A treatment during alkaline lysis does not degrade all RNA species. Since spectrophotometric measurement does not differentiate between DNA and RNA, RNA contamination can lead to overestimation of DNA concentration. RNA contamination can sometimes be detected by agarose gel analysis with routine ethidium bromide staining, although not quantified effectively. RNA bands appear faint and smeary and are only detected in amounts 2530 ng (0.5:1 RNA:DNA ratio). RNA contamination of plasmid DNA can be a concern depending on the method used for plasmid preparation. Methods using alkaline lysis with phenol extraction cannot separate RNA from plasmid DNA, leading to high levels of RNA contamination. In contrast, QIAGEN offers advanced silica-gelmembrane and anion-exchange resin technologies that ensure plasmid DNA is virtually free of RNA (79). Effect of Solvent on A260/A280 Ratio
1.90 1.85 1.80 1.75 1.70 1.65 1.60
Wa ter 1 Wa ter 2 Wa ter 3 Wa ter 4 10 mM Tris pH Cl 7.5 10 mM TrisC pH l 8.0 10 mM TrisC pH l 8.5 10 mM Tris pH Cl 9.0 50 mM Tris pH Cl 8.5 100 mM Tris pH Cl 8.5 100 mM KH 2 PO 4 pH 8.2

Figure 6. A260/A280 ratios were measured in different solvents. Red bars represent readings in ultrapure water, taken at different times after dissolution of DNA. Blue bars represent readings in various low-salt buffers as indicated.

Effect of phenol on spectrophotometric readings Phenol has an absorption maximum of 270275 nm, which is close to that of DNA. Phenol contamination mimics both higher yields and higher purity, because of an upward shift in the A260 value leading to over-quantification of DNA (Figure 7). DNA quantification by agarose gel analysis Agarose gel analysis enables quick and easy quantification of DNA (5), especially for small DNA fragments (such as PCR products). As little as 20 ng DNA can be detected by agarose gel electrophoresis with ethidium bromide staining. The DNA sample is run on an agarose gel alongside known amounts of DNA of the same or a similar size. The amount of sample DNA loaded can be estimated by comparison of the band intensity with the standards either visually (Figure 8) or using a scanner or imaging system. Influence of Phenol on Nucleic Acid UV Readings
0.4

A
Absorbance

DNA + phenol

DNA

0.0 220 Wavelength (nm) 320 3.0

0.0 220 Wavelength (nm) 320

Figure 7. Effect of phenol on quantification of DNA. A: DNA was purified on a silica membrane and split into two 100 l aliquots. 1 l of a 1:1000 dilution of phenol in water was added to one sample (final phenol dilution 1:100,000) and the UV absorbance spectrum of both samples was measured. B: UV scan of phenol, diluted 1:5000 in water.

Plasmid DNA

Absorbance

Tip

Be sure to use standards of roughly the same size as the fragment of interest to ensure reliable estimation of the DNA quantity, since large fragments interchelate more dye than small fragments and give a greater band intensity.

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More precise agarose gel quantification can be achieved by densitometric measurement of band intensity and comparison with a standard curve generated using DNA of a known concentration. In most experiments the effective range for comparative densitometric quantification is between 20 and 100 ng.

Agarose Gel Analysis of Plasmid DNA M 125 100 75 50 U ng

Tip

The amount of DNA used for densitometric quantification should fall within the linear range of the standard curve.

See A Guide to Analytical Gels, page 11, for further information on agarose gel electrophoresis.

Figure 8. An unknown amount of a 5.5 kb DNA fragment (U) was run alongside known quantities (as indicated in ng) of the same DNA fragment on a 1% TAE agarose gel. The unknown sample contained ~85 ng DNA, as estimated by visual comparison with the standards. M: 1 kb DNA ladder.

Restriction Endonuclease Digestion of DNA

Principle of restriction digestion DNA for downstream applications is usually digested with restriction endonucleases. This yields DNA fragments of a convenient size for downstream manipulations. Restriction endonucleases are bacterial enzymes that bind and cleave DNA at specific target sequences. Type II restriction enzymes are the most widely used in molecular biology applications. They bind DNA at a specific recognition site, consisting of a short palindromic sequence, and cleave within this site, e.g., AGCT (for AluI), GAATTC (for EcoRI), and so on. Isoschizomers are different enzymes that share the same specificity, and in some cases, the same cleavage pattern.

Tip

Isoschizomers may have slightly different properties that can be very useful. For example, the enzymes MboI and Sau3A have the same sequence specificities, but MboI does not cleave methylated DNA, while Sau3A does. Sau3A can therefore be used instead of MboI where necessary.

Selecting suitable restriction endonucleases The following factors need to be considered when choosing suitable restriction enzymes:

Methylation sensitivity

Fragment size

Blunt-ended/sticky-ended fragments Compatibility of reaction conditions (where more than one enzyme is used)

Fragment size Restriction enzymes with shorter recognition sequences cut more frequently than those with longer recognition sequences. For example, a 4 base pair (bp) cutter will cleave, on average, every 44 (256) bases, while a 6 bp cutter cleaves every 46 (4096) bases.

Tip

Use 6 bp cutters for mapping genomic DNA or YACs, BACs, or P1s, as these give fragments in a suitable size range for cloning.

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Blunt-ended/sticky-ended fragments Some restriction enzymes cut in the middle of their recognition site, creating blunt-ended DNA fragments. However, the majority of enzymes make cuts staggered on each strand, resulting in a few base pairs of single-stranded DNA at each end of the fragment, known as sticky ends. Some enzymes create 5' overhangs and others create 3' overhangs. The type of digestion affects the ease of downstream cloning:

Sticky-ended fragments can be easily ligated to other sticky-ended fragments with compatible single-stranded overhangs, resulting in efficient cloning. Blunt-ended fragments usually ligate much less efficiently, making cloning more difficult. However, any blunt-ended fragment can be ligated to any other, so blunt-cutting enzymes are used when compatible sticky-ended fragments cannot be generated for example, if the polylinker site of a vector does not contain an enzyme site compatible with the fragment being cloned.

Methylation Many organisms have enzymes called methylases that methylate DNA at specific sequences. Not all restriction enzymes can cleave their recognition site when it is methylated. Therefore the choice of restriction enzyme is affected by its sensitivity to methylation. In addition, methylation patterns differ in different species, also affecting the choice of restriction enzyme.

Tip Tip

Methylation patterns differ between bacteria and eukaryotes, so restriction patterns of cloned and uncloned DNA may differ. Methylation patterns also differ between different eukaryotes (see page 33), affecting the choice of restriction enzyme for construction genomic DNA libraries.

Compatibility of reaction conditions If a DNA fragment is to be cut with more than one enzyme, both enzymes can be added to the reaction simultaneously provided that they are both active in the same buffer and at the same temperature. If the enzymes do not have compatible reaction conditions, it is necessary to carry out one digestion, purify the reaction products (for example using the MinElute Reaction Cleanup Kit), and then perform the second digestion. Components of a restriction digest DNA

Water

Buffer Enzyme

DNA The amount of DNA digested depends on the downstream application. For mapping of cloned DNA, 0.21 g DNA per reaction is adequate.

Tip

DNA should be free of contaminants such as phenol, chloroform, ethanol, detergents, or salt, as these may interfere with restriction endonuclease activity.

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Enzyme One unit of restriction endonuclease completely digests 1 g of substrate DNA in 1 hour. However, supercoiled plasmid DNA generally requires more than 1 unit/g to be digested completely. Most researchers add a ten-fold excess of enzyme to their reactions in order to ensure complete cleavage.

Tip

Ensure that the restriction enzyme does not exceed more than 10% of the total reaction volume, otherwise the glycerol in which the enzyme is supplied may inhibit digestion.

Reaction volume Most digests are carried out in a volume between 10 and 50 l. (Reaction volumes smaller than 10 l are susceptible to pipetting errors, and are not recommended.)

Protocol 10. Setting up a restriction digest

Protocol 10

Tip Tip Tip

1. Pipet reaction components into a tube and mix well by pipetting. Thorough mixing is extremely important. The enzyme should be kept on ice and added last. When setting up large numbers of digests, make a reaction master mix consisting of water, buffer, and enzyme, and aliquot this into tubes containing the DNA to be digested.

2. Centrifuge the tube briefly to collect the liquid at the bottom. 3. Incubate the digest in a water bath or heating block, usually for 14 h at 37C. However, some restriction enzymes require higher (e.g., 5065C) while others require lower (e.g., 25C) incubation temperatures.

4. For some downstream applications it is necessary to heat-inactivate the enzyme after digestion. Heating the reaction to 65C for 20 min after digestion inactivates the majority of enzymes that have optimal incubation temperature of 37C.

Tip

Some restriction enzymes are not fully inactivated by heat treatment. The MinElute Reaction Cleanup Kit provides complete removal of restriction enzymes and salts following digestion.

Ligation of DNA
In order to construct new DNA molecules, DNA must first be digested using restriction endonucleases (see Restriction Endonuclease Digestion of DNA, page 18). The individual components of the desired DNA molecule are purified and then combined and treated with DNA ligase. The products of the ligation mixture are introduced into competent E. coli cells and transformants are identified by appropriate genetic selection. Appropriate control ligations should also be performed (See Protocols 1 and 2, pages 2 and 3). Removal of 5' phosphates from linearized vector DNA can help prevent vector self-ligation and improve ligation efficiency. To remove 5' phosphates from DNA, add calf intestinal phosphate (CIP) buffer and 1 U CIP and incubate for 3060 minutes at 37C. Once the reaction is complete, inactivate CIP by heating to 75C for 15 minutes.

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Protocol 11. Ligation of DNA and subsequent transformation 1. A typical ligation reaction is set up as follows:

Protocol 11

Component DNAs (0.15 g) Ligase buffer 1 l 10 mM ATP 20500 U T4 DNA ligase

2. Incubate for 124 h at 15C.

Tip Tip Tip

Simple ligations with two fragments having 4 bp 3' or 5' overhanging ends require much less ligase than more complex ligations or blunt-end ligations. The quality of the DNA will also affect the amount of ligase needed. Ligation of sticky-ends is usually carried out at 1215C to maintain a balance between annealing of the ends and the activity of the enzyme. Higher temperatures make annealing of the ends difficult, while lower temperatures diminish ligase activity. Blunt-end ligations are usually performed at room temperature since annealing is not a factor, though the enzyme is unstable above 30C. Blunt-end ligations require about 10100 times more enzyme than sticky-end ligations in order to achieve an equal efficiency.

3. Introduce 110 l of the ligated products into competent E. coli cells and select for transformants using the genetic marker present on the vector (for further information, see Protocols 1 and 2, pages 2 and 3). 4. From individual E. coli transformants, purify plasmid or phage DNAs by miniprep procedure and determine their structures by restriction mapping.

Tip

It is highly recommended to include two controls in every transformation experiment: A mock transformation without DNA. A transformation reaction with a known amount of closed circular plasmid DNA.

References 1. Sambrook, J. and Russell, D. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. 2. Ausubel, F.M. et al., eds. (1999) Current Protocols in Molecular Biology, New York: John Wiley and Sons. 3. Birnboim, H.C., and Doly, J. (1979) A rapid alkaline lysis procedure for screening recombinant plasmid DNA. Nucl. Acids. Res. 7, 1513.

Controls are essential if things go wrong. For example, colonies on plates that receive mock-transformed bacteria may indicate that the medium lacks the correct antibiotic. An absence of colonies on plates receiving bacteria transformed with plasmids under construction can only be interpreted if a positive control using a standard DNA has been included. See page 4 for further information on transformation controls.

QIAGEN offers a wide range of products for the preparation and isolation of plasmid DNA, DNA cleanup, and PCR fragment cloning (including competent cells), for all throughput and purity requirements. For further information about QIAGEN products and literature please refer to the QIAGEN Product Guide, visit us online at www.qiagen.com, or contact QIAGEN Technical Services or your local distributor.

4. Birnboim, H.C. (1983) A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100, 243. 5. Quantitation of DNA. QIAGEN News 1998 No. 2, 23. 6. Effect of pH and ionic strength on the spectroscopic assessment of nucleic acid purity. (1997) BioTechniques 22, 474. 7. QIAprep Miniprep Handbook, March 2001. 8. QIAGEN Plasmid Purification Handbook, September 2000. 9. QIAGEN Plasmid Mini Handbook, March 1999.

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